Acute deletion of mTORC1/Raptor induces thymic atrophy.

<p><b>a.</b> Age-dependent decline of mTORC1 activity in TECs. Overlaid histograms show intracellular S6 phosphorylation in EpCAM⁺CD45⁻ TECs from C57BL6/J mice of indicated ages gated as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002370#pbio.1002370.s002" target="_blank">S1A Fig</a>. Data shown are representative of three experiments. <b>b.</b> Thymus size. For b to l: Thymic atrophy after acute systemic deletion of mTORC1. Six- to 8-wk-old <i>Rpt</i>^<i>f/f</i><i>-ERCre</i> and <i>Rpt</i>^<i>f/f</i> mice were treated with tamoxifen on days 1, 2, and 5 and were euthanized on day 8. <b>c.</b> Total thymic cellularity. Bars represent mean ± standard error of the mean (SEM) (<i>n</i> = 9). Each circle or square represents one wild-type (WT) or knockout (KO) mouse, respectively. Numerical data for this figure and all other figures are in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002370#pbio.1002370.s001" target="_blank">S1 Data</a>. <b>d.</b> Thymocyte subsets. Representative dot-plots of CD4 and CD8 staining of thymocytes are shown. The gating strategy is shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002370#pbio.1002370.s002" target="_blank">S1B Fig</a>. <b>e.</b> Bar graphs represent mean ± SEM of percentages (left panel, <i>n</i> = 6) and absolute numbers (right panel, <i>n</i> = 5) of indicated thymocyte populations. <b>f.</b> Annexin-V and 7-AAD staining of DP thymocytes. The gating strategy is shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002370#pbio.1002370.s002" target="_blank">S1C Fig</a>. <b>g.</b> Hematoxylin and eosin (H&E) staining of thymic thin sections. <b>h.</b> Thymic cortex and medulla distribution revealed by immunofluorescence microcopy. Thymus cryosections were stained with primary rabbit anti-KRT5 and rat anti-KRT8 antibodies followed by secondary Rhodamine-labeled donkey anti-rabbit and fluorescein isothiocyanate (FITC)-labeled goat anti-rat antibodies. <b>i.</b> Dot-plot of TECs stained with anti-EpCAM and anti-CD45 antibodies. The gating strategy is shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002370#pbio.1002370.s002" target="_blank">S1D Fig</a>. <b>j.</b> Scatter plots represent mean ± SEM (<i>n</i> = 4) of EpCAM⁺CD45⁻ TEC percentages (left panel) and numbers (right panel). <b>k.</b> Distribution of UEA-1⁺Ly51⁻ mTECs and UEA-1⁻Ly51⁺ cTECs in gated CD45⁻EpCAM⁺ cells. The gating strategy is shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002370#pbio.1002370.s002" target="_blank">S1D Fig</a>. <b>l.</b> Percentages (left panel) and numbers (right panel) of mTECs and cTECs (<i>n</i> = 4). Data shown are representative of or calculated from at least three experiments. *<i>p</i> < 0.05; **<i>p</i> < 0.01; ***<i>p</i> < 0.001 determined by two-tailed Student’s <i>t</i> test.